RNA from cell lines and EVs was obtained by automated Maxwell RSC-Promega extractor, using, respectively, the Maxwell RSC miRNA Tissue Kit miRNA (CAT # AS1460, Promega) and Maxwell RSC miRNA Plasma and Serum kit (CAT N.AS1680, Promega).
Retro-transcription for gene detection was performed using High-Capacity cDNA Reverse Transcription Kit. For miRNA, a miRNA-specific RT was carried out using TaqMan™ MicroRNA Reverse Transcription Kit. TaqMan Individual microRNA assays (Cat. N.4427975, Thermo Fisher Scientific) were used to assess expression of hsa-miR-200a (Assay ID:001011), hsa-miR-200c (Assay ID: 000505), of hsa-miR-9 (Assay ID: 000583), hsa-miR-222 (Assay ID: 002276), has-miR-221 (Assay ID: 000524) and U6 snRNA (Assay ID: 001973). TaqMan gene expression assays (Cat. N. 4331182, Thermo Fisher Scientific) were used to assess mRNA expression of HMGB1 (Assay ID: Hs01590761g1) and ACTB (Assay ID: Hs01060665_g1). U6 and ACTB were used as normalization controls.
QPCR was performed on cDNA using Applied Biosystems ViiA 7 Real-Time PCR (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of miRNAs and mRNAs were calculated and quantified using the 2^ΔΔCq method after normalization for the expression of the controls. All procedures were performed according to the manufacturer’s instructions.
Retro-transcription for gene detection was performed using High-Capacity cDNA Reverse Transcription Kit. For miRNA, a miRNA-specific RT was carried out using TaqMan™ MicroRNA Reverse Transcription Kit. TaqMan Individual microRNA assays (Cat. N.4427975, Thermo Fisher Scientific) were used to assess expression of hsa-miR-200a (Assay ID:001011), hsa-miR-200c (Assay ID: 000505), of hsa-miR-9 (Assay ID: 000583), hsa-miR-222 (Assay ID: 002276), has-miR-221 (Assay ID: 000524) and U6 snRNA (Assay ID: 001973). TaqMan gene expression assays (Cat. N. 4331182, Thermo Fisher Scientific) were used to assess mRNA expression of HMGB1 (Assay ID: Hs01590761g1) and ACTB (Assay ID: Hs01060665_g1). U6 and ACTB were used as normalization controls.
QPCR was performed on cDNA using Applied Biosystems ViiA 7 Real-Time PCR (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of miRNAs and mRNAs were calculated and quantified using the 2^ΔΔCq method after normalization for the expression of the controls. All procedures were performed according to the manufacturer’s instructions.